Over the course of the last several years, we have found that vairous redox related species such as nitric oxide play a major role in the promotion of angiogenesis as well as part of the inflammatory response. A recent study has shown that NO regulates a number of proteins such as TSP-1, TGF beta, metallo matrix proteases (MMP), TIMP, HIF-1alpha, Akt, ERK and p53. We have found a correlation been Akt and Nitric oxide synthase in breast cancer patients. We have begun to correlate our molecular studies in vitro with patient slides. These have begun to provide us insight into the role this small diatomic radical associated with oxidative stress plays in cancer. One of the important extension of our MMP and TIMP-1 work is that we have found that changes in TIMP-1 have major effects in both Alzhiemer?s disease (AD) and Breast cancer. In breast cancer, we found that TIMP-1 predicts poor outcome and increases the association of iNOS with Akt-P. Using in vitro studies we find that TIMP-1 nitration leads to increase binding to CD63 on the tumor cell and increase Src and PI3K activity. In contrast, we find in AD that TIMP1 regulation by NO in the microglia is important for increase MMP9 activity and clearance of the plaque. This contrary behavior in these diseases indicates the importance of knowing the cell type and the molecular and chemical biological mechanism of the disease form which platform can be built to identify new markers and therapies. In animal models, we have found NOS inhibitors can induce a tumor re-growth delay after radiation thus improving the efficacy of these treatments. In collaboration with Dr. Wiltrout and Trincheri we have found that nitric oxide modification after ionizing radiation mediates strong immune response that either polarizes the immune system in a Th1 or Th2 manner. Though NOS inhibition is thought to be associated only with angiogenesis, we have found that this is a minor event. In fact, modulation of nitric oxide and other redox inflammation leads to increase Th1 cytokines such as IL-2 and IL-12 leading to an improve response. These new finding make it possible to generate these Th1 cytokines in situ thereby avoiding systemic administration. We have established the metastatic 4T1 TNBC radiation-based platform to test combined effects of immunotherapy for their ability to improve radiation therapeutic efficacy through altered redox and/or immune signaling. Our first goal will examine the influence of immune stimulants as well as NO and redox modulators during immune response to conventional radiation therapy. Improved therapeutic efficacy compared to control(s) will be determined by tumor growth delay, reduced metastatic burden, and increased median survival of treated mice. Our second goal will elucidate mechanisms associated with radiation-immunotherapy combinations. In collaboration with several groups, we are exploring radiation-immunotherapy combined effects; we propose to examine mechanisms of treatment efficacy and/or the development of resistance by evaluating the number and activation status of tumor infiltrating lymphocytes and myeloid cells as well as altered immune tolerance biomarker expression and metabolism. Our third goal will employ 3D imaging coupled with immunohistochemistry to explore spatial and temporal profile modulation of the TME in response to therapies. This information will provide 3D images of cell-cell interactions and communication networks within the TME in response to therapies. Importantly, these temporal and spatial profiles will provide key mechanistic information regarding therapeutic efficacy and/or the onset of treatment failure.